Biological Phosphoryl-Transfer Reactions: Understanding

BI80CH28-Herschlag ARI 16 May 2011 13:12

Biological Phosphoryl-Transfer Reactions:Understanding Mechanismand CatalysisJonathan K. Lassila,1,∗ Jesse G. Zalatan,2,∗

and Daniel Herschlag1

1Department of Biochemistry, Stanford University, Stanford, California 94305;email: [email protected], [email protected] of Molecular and Cellular Pharmacology, University of California,San Francisco, California 94158; email: [email protected]

Annu. Rev. Biochem. 2011. 80:669–702

First published online as a Review in Advance onApril 21, 2011

The Annual Review of Biochemistry is online atbiochem.annualreviews.org

This article’s doi:10.1146/annurev-biochem-060409-092741

Copyright c© 2011 by Annual Reviews.All rights reserved

0066-4154/11/0707-0669$20.00

∗Contributed equally to this work.

Keywords

transition state, enzymes, linear free energy relationship, kineticisotope effects, phosphate ester

Abstract

Phosphoryl-transfer reactions are central to biology. These reactionsalso have some of the slowest nonenzymatic rates and thus require enor-mous rate accelerations from biological catalysts. Despite the centralimportance of phosphoryl transfer and the fascinating catalytic chal-lenges it presents, substantial confusion persists about the propertiesof these reactions. This confusion exists despite decades of researchon the chemical mechanisms underlying these reactions. Here we re-view phosphoryl-transfer reactions with the goal of providing the readerwith the conceptual and experimental background to understand thisbody of work, to evaluate new results and proposals, and to apply thisunderstanding to enzymes. We describe likely resolutions to some con-troversies, while emphasizing the limits of our current approaches andunderstanding. We apply this understanding to enzyme-catalyzed phos-phoryl transfer and provide illustrative examples of how this mechanis-tic background can guide and deepen our understanding of enzymesand their mechanisms of action. Finally, we present important futurechallenges for this field.

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Contents

INTRODUCTION . . . . . . . . . . . . . . . . . . 670MECHANISTIC POSSIBILITIES . . . 671

Concerted Versus StepwiseReactions . . . . . . . . . . . . . . . . . . . . . . . 671

Two-Dimensional ReactionCoordinate Diagrams and aContinuum of TransitionStates . . . . . . . . . . . . . . . . . . . . . . . . . . 674

TRANSITION STATES FORPHOSPHORYL TRANSFER . . . . . 677Linear Free Energy Relationships . . . 677Kinetic Isotope Effects . . . . . . . . . . . . . 683Other Tools . . . . . . . . . . . . . . . . . . . . . . . 685Overview of Nonenzymatic Data . . . 687

ENZYMATIC PHOSPHORYLTRANSFER . . . . . . . . . . . . . . . . . . . . . . 687Activation of the Nucleophile . . . . . . . 687Stabilization of the Leaving

Group . . . . . . . . . . . . . . . . . . . . . . . . . . 690Interactions with the Phosphoryl

Oxygen Atoms: Charge,Positioning, and Geometry . . . . . . 690

Challenges for the Future . . . . . . . . . . 694CONCLUSIONS . . . . . . . . . . . . . . . . . . . . 694

INTRODUCTION

Reactions at the phosphorus atom of phosphateesters and anhydrides form the chemical basisfor many of the most fundamental processes inliving systems. These reactions allow geneticinheritance through nucleic acids and the cou-pling of chemical energy to drive the thermo-dynamically unfavorable processes required forconstruction and maintenance of living cells.Phosphoryl-transfer chemistry is also crucialfor basic metabolic pathways and cellular sig-nal transduction (1–3).

Despite the importance of phosphoryltransfer, the modern reader faces a consider-able challenge in approaching the literature onphosphoryl-transfer chemistry. In the 30 yearssince Jeremy Knowles’s major review for thegeneral reader in this series (1), many new ob-

servations have been made, some long-standingquestions have been settled, and new contro-versies have arisen. An explosion of structuraland computational data has led to new insights,but also new sources of controversy and confu-sion over chemical and catalytic mechanisms.Decades of work and often-unfamiliar exper-imental approaches can present a dauntingchallenge to investigators new to the field. Atthe same time, our current understanding ofbiological catalysis of phosphoryl transfer isclosely linked to knowledge of mechanismsand transition states for the nonenzymaticreactions in solution. In this review, we hope tocomplement excellent reviews in the chemicalliterature (e.g., 4–11) and provide the back-ground for the general reader to understandkey experimental data and concepts, to evaluatecurrent controversies in the literature, and tomeet future challenges in the field. We haveincluded additional discussion in the Supple-mental Text (for all Supplemental Material,follow the link on the Annual Reviews homepage at http://www.annualreviews.org), aswell as several compiled data sets that we hopewill be useful in future work.

Because of their biological importance, thereactions of monosubstituted phosphates (e.g.,phosphate monoesters such as glucose and inos-itol phosphate, and phosphoanhydrides such asATP and pyrophosphate) and diesters, such asDNA and RNA, are emphasized. Phosphate tri-esters and other compounds are also discussed,as they provide important mechanistic com-parisons (Figure 1). To understand how thesecompounds react and how enzymes catalyzetheir reactions, we consider four key questions:

1. Are phosphoryl-transfer reactionsconcerted, or do they proceedthrough stable intermediates? Thisquestion has largely been resolved fornonenzymatic reactions. We review theexperiments and analyses that led to itsresolution for monoesters, as this knowl-edge helps in assessing new situations,particularly in enzymatic systems wherethe question continues to arise.

670 Lassila · Zalatan · Herschlag

Supplemental Material

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http://arjournals.annualreviews.org/article/suppl/10.1146/annurev-biochem-060409-092741?file=bi-80-lassila.pdf



OP

O

–O

O–

OHP

O

O–

ORP

O

–O

O–

ORP

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–O

OR'

ORP

O

R''O

OR'

OP

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–O

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OP

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O–

OP

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N

N

N

NH2

N

O

OHOH

HHHH

ORS

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–O

O

ORP

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–O

S–

N+P

O

–O

O

N+P

O

O–

O–

–O

Thio-substituted monoestersPhosphoanhydridesPhosphate esters Phosphorylated pyridines

Monoester

Diester

TriesterAdenosine triphosphate

Pyrophosphate

Sulfate monoester

Phosphorothioate monoester Phosphorylated 3-methoxypyridine

Phosphorylated 4-methylpyridine

Figure 1Substituted phosphates and related compounds important for biology and mechanism. Phosphate monoesters and diesters form crucialbiological compounds. Phosphoanhydrides contain one or more anhydride linkages between phosphate groups. Triesters,thio-substituted compounds, and phosphorylated pyridines have contributed significantly to mechanistic understanding. Theprotonation states shown are the dominant forms at pH 7–8.

2. How can we evaluate the structureand properties of the transition state?Transition-state theory defines catalysisas preferential stabilization of a transitionstate relative to the ground state (12–15).As the proficiency of an enzyme is re-lated to its ability to recognize and sta-bilize the transition state, characterizingthe geometry, bonding, and charge dis-tribution of the transition state is crucialfor understanding catalysis. We aim to ex-plain the experimental approaches usedto characterize phosphoryl-transfer tran-sition states, providing the reader withthe tools to critically evaluate existing andemerging data.

3. Do enzymes alter transition statesfrom those in solution? If enzymes rec-ognize and stabilize the transition statesof the corresponding uncatalyzed reac-tions, then nonenzymatic transition statesmay be used as a guide to understand andpossibly engineer stabilizing interactions.Alternatively, if enzymes alter transitionstates from those in solution, it will be fas-cinating to unravel the forces and interac-tions that are responsible for the change.Although much remains to be done incomparing enzymatic and nonenzymatic

transition states, emerging data suggest acoherent picture.

4. How do phosphoryl-transfer enzymesachieve catalysis? Decades of intensivestudies have provided detailed structuralpictures and identified key enzymaticfunctional groups. Yet in most cases, theenergetic contributions of enzymatic in-teractions to catalysis remain poorly ac-counted for or unknown. We present aperspective on mechanisms for catalyzingphosphoryl-transfer reactions, selectedcase studies that illustrate key principles,and future challenges and new directions.

MECHANISTIC POSSIBILITIES

Concerted Versus Stepwise Reactions

One of the most confusing issues faced by themodern reader venturing into the mechanisticliterature is whether phosphoryl-transfer reac-tions in solution are concerted or whether theyproceed through stepwise processes with dis-crete intermediates. Although Knowles’s 1980review (1) was written more than two decadesafter seminal mechanistic results were obtained,it was still unclear at that time whether thenonenzymatic hydrolyses of phosphate mo-noesters were concerted or stepwise reactions.

www.annualreviews.org • Biological Phosphoryl Transfer 671

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Leaving group: thepart of a molecule thatdeparts in a reaction,breaking a bond tophosphorus and takinga pair of electrons

LFER: linear freeenergy relationship

KIE: kinetic isotopeeffect

Nucleophile: aspecies that donates anelectron pair to anelectrophilic atom(such as phosphorus),forming a new bond

While the issue is largely resolved today, con-fusion persists in the literature. It is instructiveto review the original data that motivated earlymechanistic models, followed by the resolutionof this issue from new data and a new conceptualframework.

Hydrolysis reactions of phosphate mo-noesters (Figure 2a) were the subject of inten-sive study for decades. As early as the 1950s,investigators observed that the hydrolysis rateincreases by many orders of magnitude asthe pH is decreased, implying that protonatedphosphate monoesters (the monoanionic form)(Figure 2b) react much faster than do phos-phate monoester dianions (16–18). To accountfor this observation, Westheimer proposed amechanism in which the rate-determining stepin phosphate monoester hydrolysis was uni-molecular decomposition to a metaphosphateintermediate, followed by rapid addition of wa-ter to produce inorganic phosphate (Figure 2c).This model explained the enhanced reactivityof phosphate monoester monoanions becausethe proton could shift from the phosphorylgroup to the leaving group, thereby stabilizingcharge buildup on the leaving group in the rate-determining step (Figure 2d).1 An intuitivelyattractive feature of this model was that the neg-atively charged oxygen atoms could act as the“driving force” for leaving-group expulsion bydonating electron density to phosphorus (18).An alternative model to explain the reactivityof phosphate monoester monoanions, in whichprotonation increased the electrophilicity of thephosphoryl group, could be ruled out becausethe monoester monoanion reacts significantlyfaster than the corresponding phosphate diester(Figure 1) (17, 18).

Although metaphosphate appeared to be toounstable to isolate and characterize, additional

1Later studies demonstrated that, for phosphate monoesterswith very good leaving groups, the dianion reacts faster thanthe monoanion does (19, 20). These leaving groups have lowpKa values, indicating that they are less prone to protona-tion, which allows the dianion reaction pathway to becomefavored.

data were taken as supporting this mechanism.Entropies and volumes of activation were citedin support of unimolecular decomposition tometaphosphate (19–21), and linear free energyrelationships (LFERs) and kinetic isotopeeffects (KIEs) suggested extensive breakingof the bond to the leaving group (19, 20, 22).We explain these techniques in later sections;here we simply emphasize that these resultswere consistent with the proposed reactionmechanism proceeding through a metaphos-phate intermediate. However, the key flawin this logic was that all of the data wereobtained from kinetic methods that reportedon properties of the transition state, not theproperties of an intermediate. Although thedata were consistent with a metaphosphateintermediate, they did not require one, and aswe describe below, considerable data availabletoday provide strong evidence for reactionsthrough metaphosphate-like transition statesbut not metaphosphate intermediates.

The distinction between a transition stateand an intermediate is apparent from reactioncoordinate diagrams (as in Figure 2e). A tran-sition state represents a local maximum alonga reaction coordinate, whereas an intermediateexists in a local minimum, i.e., an energy well.Most importantly, kinetic data such as rate con-stants and activation parameters report on theproperties of the transition state relative to thereactants (�G‡) (Figure 2e). Thus, kinetic datathat suggest significant breaking of the bondto the leaving group and minimal bonding tothe nucleophile do not necessarily imply theexistence of a metaphosphate intermediate, butrather tell us about properties of the transitionstate. How did this distinction cause decades ofconfusion in the literature? One problem wasthat the experimental data were challenging tointerpret, but a key factor was the absence ofa language or framework for describing theseand other reactions. In the next section we de-scribe a simple framework that clarifies thesepoints and helps to frame the remainder of ourdiscussion and evaluation of the mechanisms ofphosphoryl-transfer reactions.


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ΔG

Reaction coordinate

Reactants

ΔG

‡

Intermediate

Transitionstate

Products

P

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OR–O

–O

a Phosphate monoester hydrolysis

P

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HOO–

O–ROH

c Proposed mechanism for monoester dianion hydrolysis with a metaphosphate intermediate

P

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–OP

O

HOO–

O–ROH

Slow Fast

P

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O–O

Metaphosphate

RO–

d Proposed mechanism for monoester monoanion hydrolysis with a metaphosphate intermediate

P

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ORHO

–OP

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HOOH

O–ROH

Slow* Fast

P

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O–O

Metaphosphate

ROHP

O

OR–O

–OH

*This step is accelerated by protonation of the leaving group

b Phosphate monoester dianions become protonated at low pH

P

O

OR–O

–OH+ P

O

ORHO

–O

Monoesterdianion

Monoestermonoanion

e Reaction coordinate diagram

pKa ~ 6-7

H2O

H2O

H2O

Figure 2Phosphate monoesters and proposed hydrolysis reaction mechanisms. (a) Hydrolysis of phosphatemonoester dianion. (b) At low pH, phosphate monoesters become protonated to form monoanions (104).(c,d ) The rapid hydrolysis of these phosphate monoester monoanions led early researchers to propose thatphosphate ester hydrolysis reactions proceeded through a metaphosphate intermediate. There is now strongevidence against the intermediacy of metaphosphate. (e) Reaction coordinate diagram. A transition state (‡)represents a local maximum along a reaction coordinate, whereas an intermediate exists in an energy well.


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Dissociativemechanism: astepwise elimination-addition mechanismwith a stablemetaphosphateintermediate

Concertedmechanism:a single-step reactionproceeding through asingle transition state

Associativemechanism:a stepwise addition-eliminationmechanism with astable pentacoordinateintermediate

Tight transitionstate: the phosphorusatom has more netbonding to the leavinggroup and nucleophileatoms than in theground state

Loose transitionstate: the phosphorusatom has less netbonding to the leavinggroup and nucleophileatoms than in theground state

Synchronoustransition state: thetotal bond order of thephosphorus atom tothe leaving group andnucleophile atoms issimilar to that in theground state

Two-Dimensional ReactionCoordinate Diagrams and aContinuum of Transition States

Figure 3a shows a range of hypotheticalphosphoryl-transfer reaction mechanisms: adissociative mechanism (elimination-addition),which for phosphoryl transfer would entaila metaphosphate intermediate; a concertedmechanism, an SN2-type reaction proceedingthrough a single transition state with simul-taneous breaking of one bond and formationof a new bond; and an associative mechanism(addition-elimination), which would entailformation of a pentavalent phosphoraneintermediate. These reaction mechanisms,however, are only the limiting cases amonga continuum of possibilities in which bondformation and cleavage in the transition stateneed not be synchronous (Figure 3b).

To depict this continuum of possible tran-sition states, we introduce two-dimensionalreaction coordinate diagrams, also knownas More O’Ferrall-Jencks diagrams after theoriginators of this framework (23, 24). In thetwo-dimensional reaction coordinate diagramshown in Figure 3c, the reactants are depictedin the lower left corner and the products inthe upper right. Bond breaking proceeds alongthe x-axis and bond formation proceeds alongthe y-axis. This two-dimensional reactioncoordinate defines a three-dimensional freeenergy surface in which the free energy axis isperpendicular to the page. Starting from the

reactants located in a free energy well at thebottom left, a reaction will proceed across thissurface via the pathway with the lowest barrier.As in the one-dimensional reaction coordinateshown in Figure 2e, the transition state is amaximum along the reaction pathway, but aminimum in the direction perpendicular to thereaction pathway (i.e., it is a saddle point). Thetwo-dimensional reaction coordinate diagramillustrates that there is a continuum of possiblereaction pathways between the two extremes ofan elimination mechanism via a metaphosphateintermediate in the lower right corner ofFigure 3c and an addition mechanism via aphosphorane intermediate in the upper leftcorner.

The range of possible transition states forconcerted reactions can be mapped onto thereaction coordinate diagram. Transition statesin which the phosphorus atom sees an increasein the total bond order to the nucleophileand the leaving group relative to that in thereactants are referred to as tight transitionstates. Those with a decrease in total bondorder between phosphorus and the nucleophileand leaving group are called loose transitionstates. Concerted reactions with similar netbond order between reactants and transitionstates are synchronous transition states (for amore sophisticated treatment of bond order,see Reference 25). These transition states aredepicted as symmetric in Figure 3c but neednot be—the nucleophile and leaving group may

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 3Mechanistic possibilities in phosphoryl-transfer reactions. (a) An elimination-addition reaction through ametaphosphate intermediate (DN + AN), an addition-elimination reaction through a phosphoraneintermediate (AN + DN), and a concerted reaction pathway with simultaneous bond formation and bondcleavage (ANDN). The parentheses give the IUPAC nomenclature (162). (b) A range of possible transitionstates for a concerted pathway. (c) A two-dimensional reaction coordinate diagram, also known as a MoreO’Ferrall-Jencks diagram (23, 24). The diagram represents a range of possible concerted reaction pathwayspassing through loose, synchronous, or tight transition states. Bond breaking and bond formation proceedalong the x- and y-axes, respectively, and the energy axis is perpendicular to the page. The transition state islocated at a maximum along the path from reactants to products, but a minimum in the directionperpendicular to the reaction pathway. The two-dimensional reaction coordinate depiction emphasizes thatthere is a continuum of reaction pathways, and the transition state can occur at any point along the pathway.For simplicity, the diagram above depicts symmetrical transition states halfway along each reaction pathway.Reactions proceeding through intermediates would have additional energy wells in the lower right corner fora metaphosphate intermediate and in the upper left corner for a phosphorane intermediate.


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Synchronous

Lo

ose

Tight

Reactants

Products

Bond breakage

Bo

nd

fo

rma

tio

n

‡

‡

‡

a Hypothetical reaction mechanisms for phosphoryl transfer

b A range of possible transition states for a concerted pathway

c Two-dimensional reaction coordinate diagram

P

O

OR

OO

NuORP

O

NuO

O

P

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ORO

OP

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NuO

O

P

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ORO

ONu

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Nu OR

OR

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OP

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OO

ORP

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NuO

O

P

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OR

OO

NuP

O

ORO

ONu

P

Oδ−

OR

OO

Nu

δ−δ−

δ−δ+P

O

ORO

ONu ORP

O

NuO

O

P

O

OR

OO

Nu

δ−

δ−δ−

δ−δ+

P

Oδ−

OR

Oδ−δ−O

Nuδ−δ+

Elimination-addition reaction (dissociative)

Metaphosphate intermediate

Phosphorane intermediate

TIGHT transition state

LOOSE transition state

Addition-elimination reaction (associative)

Concerted reaction

Nu Nu OR


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TERMINOLOGY FOR DESCRIBINGPHOSPHORYL-TRANSFER REACTIONS

Here we use the terms loose, synchronous, and tight to de-scribe different types of transition states for a concerted reaction(ANDN). In the literature, the terms “associative” and “dissocia-tive” have often been used to describe transition states. This usageis ambiguous because the terms have been used to refer to both themechanism of the reaction (whether concerted or stepwise) andthe structure of the transition state in a concerted reaction. Thus,the following terminology has been suggested and is used herein.An associative, or addition-elimination (AN + DN), mechanismis a reaction that proceeds through a phosphorane intermediate.A dissociative, or elimination-addition (DN + AN), mechanism isa reaction that proceeds through a metaphosphate intermediate.The parentheses give the corresponding IUPAC nomenclature(162).

be bonded to phosphorus to differing extentsin transition states that are early or late alongthe reaction pathway. For further discussion ofterminology, see the sidebar Terminology forDescribing Phosphoryl-Transfer Reactions.

For phosphate monoester hydrolysis reac-tions, a metaphosphate-like transition state canoccur for a reaction pathway that proceeds nearthe bottom right corner, but this pathway neednot proceed through a metaphosphate inter-mediate. Indeed, there is now strong evidenceagainst the formation of a metaphosphateintermediate in phosphate monoester hydrol-ysis (26–33). Most simply, stereochemicalstudies demonstrate that hydrolysis proceedswith complete inversion of configuration (26,27). This result rules out the presence of along-lived, freely diffusing metaphosphateintermediate, because water could attack a freemetaphosphate from either side to produce aracemic mixture of products (for a discussion ofsome specific types of reactions where racem-ization is observed, see Supplemental SectionB). However, metaphosphate could still form,but not be long-lived enough to diffuse andracemize; such reactions are sometimes re-ferred to as preassociation stepwise (34). Strongevidence against even transient formation of

a metaphosphate intermediate came laterfrom LFERs demonstrating that phosphoryl-transfer reactions are sensitive to the strengthof a series of amine nucleophiles (28–32). Thekey feature of these experiments was the use of abroad range of nucleophile strengths such thata change in the rate-limiting step would havebeen detected if an intermediate occurred alongthe reaction pathway (29–32). Corresponding,although not identical, logic was subsequentlyapplied to reactions of oxygen nucleophiles,including water, strongly suggesting thatmetaphosphate is not an intermediate inphosphate monoester dianion hydrolysis (33).Similarly, LFERs, stereochemical studies, andKIEs suggest that hydrolysis of phosphatemonoester monoanions (Figure 2b) proceedsthrough a loose transition state and provideno indication of formation of a metaphosphateintermediate (20, 26, 35, 36). The interestedreader should consult the original referencesafter having read the following sections, whichintroduce the key concepts for understandingthese LFER experiments.

As for monoesters, experimental datasuggest that phosphate diesters and triesters(Figure 1) generally react through concertedprocesses (37, 38), although there are notableexceptions where stable phosphorane interme-diates have been observed (discussed furtherin Supplemental Section B). The ability tocompare reactivity trends among phosphatemonoesters, diesters, and triesters has providedan important perspective for interpretingexperimental data about transition-state prop-erties. The experimental evidence supportsmonoester reactions proceeding through loosetransition states, and the transition states tendtoward increasing tightness from monoesterto diester to triester. The next sections explainhow experimental methods can allow us toinfer properties of the transition state andindirectly assess the extent of bond cleavageand bond formation. Because enzymatic rateenhancements arise from preferential stabi-lization of the transition state relative to theground state (12–14, 39), characterizing thestructure of the transition state is a critical step



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toward understanding how enzymes producetheir enormous rate enhancements.

TRANSITION STATES FORPHOSPHORYL TRANSFER

No experimental methods yet exist for directlyvisualizing transition states for phosphoryl-transfer or other reactions in solution. How-ever, kinetic data can provide indirect but pow-erful information about transition states, andsystematic variation of reactants and conditionscan allow more incisive feedback about theproperties of the transition state. Here we re-view fundamental experimental approaches forcharacterizing phosphoryl-transfer reactions,emphasizing both the information obtained andthe limitations of each approach.

Linear Free Energy Relationships

Analyses of substituent effects have been anessential part of the development of our mod-ern understanding of organic reactivity (15,40–43). By systematically altering the structureof a molecule and measuring the resultingeffects on reaction rates and equilibria, one cancharacterize the transition states for chemicalreactions. The idea is simple and intuitive: Ifa bond is broken or formed in the transitionstate, there will be a change in electron densityand charge at that position in the progressionfrom reactants to the transition state. Addingelectron-withdrawing groups stabilizes devel-oping negative charge and destabilizes develop-ing positive charge, whereas adding electron-donating groups has the opposite effect (40, 42).

This concept is illustrated in Figure 4 withthe phenolate leaving group of phenyl phos-phate as an example. In the hydrolysis reac-tion of phenyl phosphate dianion, the P-Obond to the phenolate leaving group is bro-ken, yielding a phenolate with a net charge of−1. In the transition state when the P-O bondis partially cleaved, charge has developed onthe phenolate oxygen atom, but to a lesser ex-tent than in the phenolate product (Figure 4a).The extent of charge buildup in the transition

state relative to the products corresponds tothe extent of bond cleavage in the transitionstate. To assess charge buildup in the transi-tion state, electron-withdrawing and electron-donating groups are added to the leaving group.For example, adding a nitro group to the pheno-late leaving group withdraws electron density,stabilizing the charge development in the tran-sition state and increasing the hydrolysis rateconstant (Figure 4b). The degree to which thenitro group increases the rate constant dependson how much negative charge develops in thetransition state of the reaction. In a loose tran-sition state with extensive bond cleavage to theleaving group, there is a lot of charge devel-opment so electron-withdrawing groups givelarge rate increases. In contrast, in a very tighttransition state with little bond cleavage to theleaving group, electron-withdrawing groupswill have little or no effect on the reaction rate.

What does it mean to say that electron-withdrawing groups give large or small rateincreases for reactions involving extensive orminimal bond cleavage? To quantitativelyinterpret the significance of an observed rateeffect, it is also necessary to measure the effecton the overall equilibrium for the reaction, asthis effect sets the scale for how the substituentstabilizes complete charge buildup on theleaving group (43, 44). These comparisons areperformed using logarithms, which provide acommon scale that is directly proportional tochanges in free energy.2 In the case of phenylphosphate hydrolysis, adding an electron-withdrawing group stabilizes negative chargeon the phenolate product, shifting the overallequilibrium toward the products (Figure 4c).If the resulting effects on log(k) and log(KEQ)were the same, it would suggest that the chargedevelopment in the transition state is similarto that in the products (Figure 4a), implying

2These relationships, which can be found in standardphysical chemistry textbooks, are as follows: �G =−2.303 RTlog(KEQ) and �G = −2.303 RTlog(k) +2.303 RTlog(κkBT/h), where R is the gas constant, T is tem-perature in Kelvin, κ is the transmission coefficient, kB isBoltzmann’s constant, and h is Planck’s constant.


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+ P N+

O

O–

N+

O

O–

O+

+ P

HO

HO +

+

–OP

O

O OP

O–

––

P

O

O O

–

––

+

+ P

O

–O–O

O + –OH+

–O–O

OH +

–

log

k

log

k

log

k

Tight SynchronousLoose

pKa pKa pKa

N+

–OO–

O–

ON+

–OO–

O

N+

–OO–

O

O–O–

+ P

O

–O –O–O

–O–O

–O

O + –O

–O

–O

–O

H+P

O

OH +

+ P

O

O +H+

H+

H+

P

O

OH + N+

O

O–

N+

O

O–

N+

O

O–

N+

O

O–

Full development of negative charge

Partial development of negative charge

a Negative charge develops on phenolate oxygen in hydrolysis of phenyl phosphate dianion

b Electron-withdrawing group stabilizes developing negative charge on phenolate oxygen

c Electron-withdrawing group stabilizes phenolate product in overall equilibrium

d Electron-withdrawing group stabilizes negative charge on free phenolate

e Hypothetical leaving group LFER with different transition states

Reactants Transition state Products

H2O

O

–O–O

OH2O H2O

O+

P

O

O O

–

––

H2O

O

–O–O

OH2O

H2O

H2O

–O–O

H2O

Add nitro group

Decreased pKa

Add nitro group

Products stabilized

Add nitro group

Faster reaction

pKa = 10

pKa = 7.2


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that the P-O bond is nearly or fully broken inthe transition state. Changes in log(k) that aresmall (or zero) relative to changes in log(KEQ)suggest that the P-O bond remains mostly(or fully) intact in the transition state. Thecorrelation between log(k) and log(KEQ) is anexample of a LFER and the slope of this LFERreflects the extent of charge buildup and bondcleavage (or formation) in the transition state.

In practice, LFERs are typically reported asplots of log(k) versus pKa and log(KEQ) versuspKa rather than log(k) versus log(KEQ), wherethe pKa is that for the nucleophile or leavinggroup in the reaction of interest. The slopes ofthese so-called Brønsted plots are referred to asβNUC for a series of rate constants with differ-ent nucleophiles, βLG for a series of rate con-stants with different leaving groups, and βEQ forcomparisons of log(KEQ) versus pKa. The pKa,which is also a logarithmic quantity, provides aconvenient standard reference scale with whichto quantify the effects of electron-withdrawingand electron-donating groups. In the phenolateexample, the electron-withdrawing effect of thenitro group decreases the affinity of the substi-tuted phenolate for a proton, reducing its pKa

value (Figure 4d). If there is substantial chargedevelopment in the transition state, as in a loosetransition state, the nitro group has a large effecton rate constants relative to pKa and the βLG

slope is steep (Figure 4e). In contrast, a tighttransition state will have little charge develop-ment relative to pKa and a resulting shallow βLG

slope. In nucleophile LFERs, a loose transitionstate in which the nucleophile develops a smallamount of positive charge has a small, positiveβNUC value. A tight transition state develops

greater positive charge, and thus a larger, posi-tive value of βNUC is expected.

Comparisons to pKa values provide a usefulreference for charge development because, sim-ilarly to the overall equilibrium for bond cleav-age, the deprotonation equilibrium involves thedevelopment of a full −1 charge on the phe-nolate and because pKa values are particularlyconvenient to measure. In contrast, KEQ valuesare difficult to obtain, especially for reactionswhere one side of the equilibrium is stronglyfavored. Nevertheless, there is a drawback tousing pKa as a proxy for log(KEQ).

Changes in log(KEQ) calibrate the scale forcomplete bond cleavage (or formation) in thereaction of interest, but changes in pKa reporton a different reaction, i.e., deprotonation.The substituent effects can be different fordeprotonation and the reaction of interest (i.e.,|βEQ| �= 1) (43), which makes values of βNUC

and βLG less straightforward to interpret interms of the extent of bond cleavage. Thisproblem can be remedied by obtaining the ratioβLG/βEQ (or βNUC/βEQ), which corresponds tothe slope of the plot of log(k) versus log(KEQ).This value is sometimes referred to as theLeffler α parameter (40, 43, 44). This approachis convenient when direct measurement ofKEQ is challenging but an estimate of βEQ

is available, usually from a set of differentbut structurally related compounds. Anotherconceptual framework useful for interpretingβ values is effective charge, which is discussedin Supplemental Section C (40, 43).

In Figure 5a,b, experimental LFERs areplotted for reactions of monosubstituted phos-phoryl compounds (19, 20, 28, 31–33, 45–47)

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 4Substituent effects can provide information about charge development in the transition state. (a) In the complete hydrolysis reaction ofphenyl phosphate, the P-O bond to the leaving-group oxygen is fully broken, and negative charge develops on the phenolate oxygen. Inthe transition state, the P-O bond is partially broken, and negative charge partially accumulates on the phenolate oxygen. (b) Adding anelectron-withdrawing p-nitro group distributes electron density away from the leaving-group oxygen in the transition state, making thepartial cleavage of the P-O bond more favorable and thereby increasing the reaction rate. (c,d ) Adding a p-nitro group also withdrawselectron density in the free phenolate, shifting the overall equilibrium toward products and reducing the affinity for a proton. Thisresults in a reduced pKa value. (e) Hypothetical linear free energy relationships (LFERs) for a series of leaving groups of differing pKavalues.



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Leaving group pKa

P

O

OO

–

–

N N

–

+

P

O

N+ +P–O

–O

O

N+

X

N

X+

N

X

–12

–8

–4

0

0 4 8 12

O–

O–

N+

–O

O

–15

–10

–5

0

5

–5 0 5 10 15 20

a Leaving group LFERs for monosubstituted phosphates

c Reactions with nitrogen nucleophiles and leaving groups

b Nucleophile LFERs for monosubstituted phosphates

log

(k,

M–

1s–

1)

log

(k,

M–

1s–

1)

Nucleophile pKa

βLG = –0.95

βLG = –0.98

βLG = –1.26

βNUC = 0.16

βNUC = 0.08

βNUC = 0.1

O–O–

Figure 5Interpreting observed linear free energy relationships (LFERs). (a) Observed βLG LFERs. In gray: substituted phosphate monoesterdianions reacting with water [diamonds indicate benzoyl leaving groups (19); circles (20) and square (45) are phenolate leaving groups].Points are indicated for phenyl phosphate and p-nitrophenyl phosphate. In red: hydrolysis reactions of phosphorylated pyridines[squares (31) and circles (33)]. In blue: morpholine nucleophile reacting with phosphorylated pyridines (47). (b) Observed βNUC LFERs.In gray: amine nucleophiles reacting with p-nitrophenyl phosphate (28). In red: oxygen nucleophiles with phosphorylated 4-methylpyridine (46). In blue: amine nucleophiles with phosphorylated 3-methoxypyridine (32). (c) Pyridine nucleophiles and leaving groups donot have transferrable protons and show the same trends as for oxygen nucleophiles (panels a and b).

(see Supplemental Section D for additionaldata sets). For the hydrolysis reactions of phos-phate esters, there is a large sensitivity to leav-ing group pKa (βLG = −1.26) (Figure 5a).Using a value for βEQ of −1.35 that has been es-timated for hydrolysis reactions of monoesterswith oxygen leaving groups (48), we can esti-mate the fraction of total charge developmenton the leaving group in the transition state as|βLG/βEQ| = 0.93, suggesting that a largeamount of bond breakage to the leaving grouphas already occurred in the transition state.

To conclude that the transition states forthese reactions are loose, we must also considerbond formation, as extensive bond cleavageto the leaving group could also be observed

for a late transition state with substantialbond formation to the incoming nucleophile.To evaluate both βNUC and βLG, we turnto nitrogen nucleophiles and leaving groups(Figure 5c), as more complete data areavailable for these reactions. Here βLG =−0.95 for reaction of morpholine with phos-phorylated pyridines (47), βNUC = 0.16 forreaction of pyridines with 3-methoxypyridine(32), and βEQ has been estimated to be 1.05(Figure 5a,b) (32). These values correspond toestimated charge changes of |βLG/βEQ| = 0.90and |βNUC/βEQ| = 0.15, suggesting extensivebond cleavage to the leaving group and littlebond formation to the nucleophile, i.e., a loosetransition state in the lower right corner of


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the two-dimensional reaction coordinatediagram (Figure 3c).

The two-dimensional reaction coordinatediagram can also be used as a conceptual toolto understand how changes in structure canchange the transition state. Following is anabbreviated discussion; the reader is referredto Supplemental Section C and the originalliterature for more detailed treatments (44, 49,50). According to the Hammond postulate,two states along a reaction coordinate that areclose in free energy will have similar structures(44, 51). Consequently, perturbations that raisethe energy of either the reactants or productsresult in a shift of the transition state towardthe higher-energy species (Figure 6a). Thislogic holds along the pathway from reactantsto products, where the transition state is amaximum in free energy. Perturbations thatare not along the reaction path, such as thosethat raise the energy of either the phosphoraneor metaphosphate intermediate species, havedifferent consequences. Because the transitionstate represents a minimum in free energyalong the direction perpendicular to the reac-tion pathway (e.g., along the line between thephosphorane and metaphosphate intermediatecorners in Figures 3c and 6c), the transitionstate shifts in the direction of the stabilizedspecies. This is sometimes referred to as ananti-Hammond effect (Figure 6b) (50).

Using this logic for how the transition-statestructure changes in response to perturbationsin the reaction free energy surface, it is possi-ble to assess crudely the effect of adding ad-ditional substituents in the progression frommonoesters to diesters to triesters. Figure 6c

shows a two-dimensional reaction coordinatediagram for symmetric phosphoryl-transfer re-actions with alkoxide nucleophiles and leavinggroups. To a first approximation, adding anester substituent to the phosphoryl group hasno differential energetic effect on the reactantsand products (especially in a symmetrical reac-tion where the nucleophile and leaving groupare the same), so the transition state does notshift along the reaction pathway. In the direc-tion perpendicular to the reaction pathway, the

phosphorane corner has significant chargebuildup on the phosphoryl group, and addingan ester substituent that withdraws electronsis expected to stabilize the phosphorane cor-ner relative to the metaphosphate corner.Thus, adding ester groups is predicted to haveanti-Hammond effects on the transition state(Figure 6b), leading to a progression from looseto tighter transition states for monoesters, di-esters, and triesters (Figure 6c).

The currently available LFER data for mo-noester, diester, and triester reactions are gen-erally consistent with this expectation. Indeed,the approximate positions of the transitionstates in Figure 6c are predicted from analysesof almost 100 LFERs reported in the literature(see Supplemental Sections C and D). Re-markably, despite this large number of LFERs,there remain many gaps in the data and, corre-spondingly, in our understanding. Informationis particularly limited for symmetric reactions,which provide the simplest comparisonsbetween these reactions and their transitionstates. Still, the available data provide a consis-tent picture and make strong predictions aboutreactions that have not yet been tested.

Interestingly, phosphate monoesters showlimited variability in transition-state prop-erties, based on the available data, whereasthe variability is considerable for phosphatetriesters. Panels c and d of Figure 6 depictapproximate predicted transition-state posi-tions for symmetric reactions with alkoxidesand with p-nitrophenolate, respectively. Forthese two types of reactions, which differ inthe pKa of the nucleophile and leaving group,the position of the monoester transition stateis similar—a loose transition state in the lowerright corner. In contrast, triester reactionsshow greater variability in transition-stateproperties with pKa. The available data suggesta range of possible transition states for triestersfrom loose to tight. In all cases, the directionof change is as predicted from considerationof Hammond and anti-Hammond effects intwo-dimensional reaction coordinate dia-grams; however, the size of the change differsgreatly. The simplest expectation for this


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Hammond effect along the reaction pathway a Anti-Hammond effect perpendicular to thereaction pathway

b

c Approximate predictions for symmetric reactions withalkoxide nucleophiles and leaving groups

Reactants

Products

Bond breakage

Bo

nd

fo

rma

tio

n

‡

‡

‡

P

O–

OR

O––O

RO P

O–

OR

O––O

RO–ORP

O

ROO–

O–

Products

–ORP

O

ROO–

O–

P

O

–OR

OO

P

O

OR–O–O

Triester

Diester

Monoester

Bond breakage

Bo

nd

fo

rma

tio

n

‡

‡

‡Triester

Diester

Monoester

Adding ester groupsstabilizes phosphorane

Adding ester groupsstabilizes phosphorane

d Approximate predictions for symmetric reactions withp-nitrophenolate nucleophiles and leaving groups

RO–

Reactants

P

O

OR–O–O

RO–RO–

‡

‡

ProductsReactants

ProductsReactants

ΔG

ΔGΔG

Products

Metaphosphate

Reactants

Phosphorane

‡

(–β N

UC

/βE

Q)

(βLG/βEQ) (βLG/βEQ)

(–β N

UC

/βE

Q)

–

P

O

–OR

OO

RO–

–

‡Products

Metaphosphate

Reactants

Phosphorane

ΔG

Figure 6Changes in structure can alter the nature of the transition state. (a) Along the reaction pathway, the transition state is at a maximum infree energy. When the equilibrium between reactants and products changes, the transition state moves toward the species that hasincreased in energy. (b) Perpendicular to the reaction pathway, the transition state is at a minimum in free energy. When theequilibrium between the phosphorane and metaphosphate species changes, the transition state moves toward the species that hasdecreased in energy. (c) A two-dimensional reaction coordinate diagram for symmetric phosphoryl-transfer reactions with alkylnucleophiles and leaving groups, with approximate predicted changes in transition-state structure as ester substituents are added to thephosphoryl group and the energy of the phosphorane corner decreases. (d ) Similar approximate predictions for symmetric reactionswith p-nitrophenolate nucleophiles and leaving groups. Whereas monoester reactions show little variability in transition state, triestersshow considerable variability (as discussed further in Supplemental Section C).


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difference would be a shallower energy surfacefor triesters, but the data remain limited. Manychallenges remain to obtain more systematicLFERs and to understand the origins of thesedifferent behaviors. Supplemental Section Ccontains additional discussion.

An important potential limitation of LFERsis that alternative mechanisms and changesto the energetic landscape can complicatethe interpretation of LFERs. Indeed, theextreme view that LFERs cannot be used toevaluate reaction mechanisms has been putforth in the literature (52–54). This argumenthas been advanced in two ways. First, it hasbeen suggested that proton transfers among thenucleophile, the phosphoryl oxygen atoms, andthe leaving group could obscure meaningfultrends related to transition-state properties(52). Although this complication can arise insome reactions, this potential problem hasbeen eliminated in many of the fundamentalstudies of phosphoryl transfer through useof nitrogen nucleophiles and leaving groupssuch as pyridines that have no transferableprotons (Figure 5) (see SupplementalSection D). The transition states character-ized for these monosubstituted phosphorylcompounds are essentially indistinguishablefrom those for reactions of phosphate mo-noesters with other nitrogen and with oxygennucleophiles. These similarities suggest ananalogous mechanism for all these reactions.Finally, oxygen nucleophiles both with andwithout potentially transferable protons followa single LFER with only minor deviations,providing strong evidence against interventionand complication from proton transfers in thesereactions (33). For enzymatic reactions there isalso evidence against this class of mechanism,termed substrate-assisted catalysis, as discussedbelow in Enzymatic Phosphoryl Transfer.

A second challenge to the interpretationof LFERs is based on the suggestion thattransition states change with substituent pKa

(53, 54). However, this point has been coveredextensively in the development and theory ofLFERs (40, 49, 50, and references therein),and the experiments themselves address the

question of changes in transition state with pKa

(discussed further in Supplemental SectionC). Within a single LFER, curvature indicateschanges in transition-state bonding over therange of compounds used to construct theLFER. Similarly, changes in the slope acrossa series of LFERs (e.g., a set of βLG valuesobtained with different nucleophiles) suggestchanges in transition-state bonding, and aconstant slope suggests similar transition-statebonding. As discussed above, the data for phos-phate monoesters indicate only small changesin transition-state structure with different nu-cleophile and leaving-group pKa values (33). Incontrast, LFERs for triesters indicate changesin the nature of the transition state with differ-ent substituent pKa values (37, 38, 55). In bothcases, the directions of the changes can be un-derstood simply in terms of perturbations to theenergy surface in a two-dimensional reactioncoordinate diagram (37, 55). Thus, the LFERdata, although not complete, are consistentlyand robustly accounted for by the current andlongstanding models described above. Never-theless, it is important to recognize that LFERdata do not directly supply information aboutbond lengths and charges in the transitionstate, but rather provide empirical correlationsfrom which transition states are inferred. Moreresearch is needed to fully understand theseand other empirical relationships.

Kinetic Isotope Effects

KIEs report on changes in bonding that occurin the transition state by measuring changesin reaction rates that occur when an atom inthe compound of interest is substituted with aheavy isotope (Figure 7). How does isotopicsubstitution alter reaction rates, and how dothese changes report on properties of the tran-sition state? According to quantum mechanics,every bond has a characteristic minimum vi-brational energy state, termed the zero-pointenergy, which depends on the strength of thebond and the masses of the atoms involvedin the bond; increasing mass decreases thezero-point energy. When a bond is broken, the



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ΔG

Reaction coordinate (P-O bond cleavage)

Reactant Transition state

18O

16O

18O

16O

ΔG‡ (

16O

)

ΔG‡ (

18O

)

a

b

P

O

O–O

–O NO2

Nu P

O

O

OO

NO2Nu

δ−

δ−δ−

δ−δ+–O NO2P

O

NuO–

O–

16O/18O

Product

EP-O

EP-O

Figure 7Kinetic isotope effects (KIEs) report on changes in bonding in the transition state. (a) Sites of isotopic substitution in the phosphatemonoester p-nitrophenyl phosphate (pNPP) are shown in color. KIEs can be measured for multiple sites within a molecule, includingthe bridging oxygen at the position of bond cleavage (red ) and the nonbridging oxygen atoms of the phosphoryl group (blue). Thepresence of a nitrogen atom ( green) in pNPP allows KIEs to be measured with the remote label method (56), as it is technically easier tomeasure 15N/14N than 18O/16O ratios. Recently, however, some KIEs have been determined by direct measurement of 18O/16O ratios(10, 63, 101). (b) Heavy-isotope substitution at the position of bond cleavage (red, bridging oxygen atom) slows the reaction because thedifference in zero-point vibrational energy is larger in the ground state than in the transition state, leading to a larger activation barrierfor the heavy-isotope-substituted molecule (56, 57). The broader vibrational potential well at the transition state reflects the weakerstate of the partially broken bond.

vibrational energy states for that bond are lostso there is no longer a difference in vibrationalenergy between the heavy and light isotopes.Thus, there is a larger energy barrier for cleav-age of a heavy-atom substituted bond and thereaction is correspondingly slower; this leads toa “normal” KIE (klight/kheavy > 1) (56, 57). Forbonds that are partially broken in the transitionstate, the difference in vibrational zero-pointenergy between the heavy and light isotopes

decreases relative to that in the ground state,and the magnitude of the KIE corresponds tothe extent of bond cleavage in the transitionstate (Figure 7b). In cases where bonding in-creases at the substituted position, for exampleas a result of protonation, an inverse isotope ef-fect can be observed (klight/kheavy < 1). KIEsresulting from isotopic substitutions at posi-tions of bond cleavage or formation are termedprimary isotope effects.


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KIEs can also be observed for isotopicsubstitution at positions that are not directlyinvolved in bond cleavage or formation (i.e.,secondary isotope effects). In general, bondvibrational states include stretching, bending,and torsional modes. For primary isotopeeffects, the dominant vibrational contributiontypically comes from loss (or gain) of a stretch-ing mode involving the groups in the bondbeing broken or formed. In contrast, secondaryisotope effects frequently arise from changesin geometry, which can have significant effectson bending and torsional vibrational modes.For example, deuterium substitution in SN2displacement reactions at carbon leads to small,normal secondary KIEs due to a weaker bend-ing mode in the trigonal planar transition-stategeometry relative to the tetrahedral groundstate (57). Similar geometrical changes occurfor the nonbridging oxygen atoms duringphosphoryl-transfer reactions. Unlike deu-terium, however, the nonbridging oxygenatoms can also undergo increased or decreasedbonding to the central phosphorus atom in thetransition state, leading to changes in stretchingvibrational modes. Thus, predicting the domi-nant contribution to the KIE is not straightfor-ward. Nevertheless, trends in these KIEs canstill be informative, and we hope these valuescan be understood more fully in the future.

Because isotope substitution is minimallyinvasive and can be made at multiple posi-tions in a phosphoryl compound (Figure 7a),this approach would appear to be a power-ful method to obtain information about transi-tion states. In practice, the compounds neededare not trivial to make and the analysis re-quires high-precision measurements with so-phisticated equipment (56). Notwithstandingthese challenges, there is a large body of KIEdata for phosphoryl-transfer reactions in theliterature that provides a consistent picture ofphosphoryl-transfer transition states. This pic-ture complements information obtained fromLFER studies.

Figure 8 shows the KIEs for hydrolysisreactions of phosphate monoesters, diesters,and triesters with p-nitrophenyl leaving

groups. For comparison, leaving-group KIEsfor phosphorothioate and sulfate esters are alsoincluded, because these compounds displaysimilar behaviors as their phosphate ester coun-terparts. Leaving-group KIEs for monoestersare large and normal, consistent with a loosetransition state with significant bond cleavageto the leaving group (Figure 8a). The KIEs fordiesters and triesters are significantly smaller,suggesting tighter transition states for thesecompounds. Nonbridging oxygen atom KIEsalso exhibit a trend from monoester to triester;values for monoesters are small, whereas thosefor triesters are significantly larger (Figure 8b).This increase in the nonbridging KIE may arisebecause increasing nucleophilic participationin a tighter transition state leads to weakeningof the P-Ononbridge bond. Taken together,the KIEs suggest a trend from loose to tighttransition states for phosphate monoesters,diesters, and triesters. Although the data arelimited, leaving-group KIEs for triesters varysignificantly with the identity of the leavinggroup (58, 59), consistent with the larger bodyof LFER data suggesting that the transitionstates for triester reactions are highly variable.A detailed compilation of LFER and KIE datafor phosphoryl-transfer reactions supportingthese general conclusions is presented inSupplemental Sections C, D, and F.

Because isotope effects are sensitive tochanges in both bond lengths and geometry,and more generally to any changes in the vibra-tional environment of the labeled position (57),KIEs can be challenging to interpret in complexenvironments. This challenge is particularlyacute in the active site of an enzyme, where pro-tonation, metal ion interactions, and interac-tions with other enzyme functional groups cancontribute to observed isotope effects (56, 60–63). KIEs for representative enzyme-catalyzedreactions are provided in SupplementalSection F.

Other Tools

Measurements of volumes and entropies of ac-tivation have been consistent with the trends


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PhosphateK

IE

KIE

PhosphorothioateSulfate

1.030

1.025

1.010

1.005

1.015

1.020

0Monoesters Diesters Triesters

1.010

1.005

0

0.995Monoester Diesters Triester

Leaving group KIEsa b Nonbridging oxygen atom KIEs

Figure 8Kinetic isotope effects (KIEs) for nonenzymatic phosphoryl-transfer reactions with p-nitrophenyl leaving groups (35, 59, 163–166)(includes one unpublished value from E.A. Tanifum & A.C. Hengge). Complete data tables and corresponding references are presentedin the Supplemental Section F. The reactions shown are for the fully ionized species (i.e., phosphate monoester dianions and sulfatemonoester monoanions, rather than the corresponding protonated forms). (a) Leaving-group KIEs (18kbridge) for monoester hydrolysisare large, suggesting extensive bond cleavage to the leaving group in the transition state. Values of 18kbridge are significantly smaller fordiesters and triesters. Phosphorothioate and sulfate esters exhibit similar trends in their leaving-group KIEs. (b) The nonbridgingoxygen atom KIE (18knonbridge, per oxygen atom) for phosphate monoester hydrolysis is close to unity, whereas that for triesterhydrolysis is large and normal. Values for diester hydrolysis are more variable. Phosphorothioate esters are not included because thepresence of sulfur at the nonbridging position significantly perturbs 18knonbridge (see Supplemental Section F), and sulfate esters areomitted because 18knonbridge is available for only a single sulfate monoester.

from LFER and KIE data, suggesting loosetransition states for monoesters and tightertransition states for diesters and triesters. How-ever, a serious concern in interpreting thesedata is the difficulty of isolating changes involume or entropy of the reacting systemfrom changes due to interactions with the sol-vent. We present and discuss these data inSupplemental Section G.

Thio-substitution experiments have beenan important tool in studying phosphoryltransfer. Substitution of a sulfur atom for anoxygen atom of a phosphate ester leads to per-turbation in pKa values, bond lengths, van derWaals radii, polarizability, and electrostaticsof the resulting compound, providing valuablemechanistic comparisons in catalyzed reactions(64–67). For example, catalytic metal interac-tions in enzymes can be probed with “metal ionrescue” experiments, in which loss in activityupon thio-substitution can be reversed throughuse of thiophilic metal ions (68–72). “Sulfurrescue” experiments, in which deleterious

mutations are rescued by the lowered pKa

values of thio-substituted substrates, can helpto identify general acid catalysis (67, 73, 74).Thio-substitution at prochiral oxygen positionscan help to identify the stereochemical courseof reactions, and within the context of an activesite, rate effects due to steric differences uponthio-substitution can provide probes for active-site interactions and geometry (75–78). Anunderstanding of the nonenzymatic reactivitiesand properties of thio-substituted compounds,also called phosphorothioates (with nonbridg-ing sulfur atom) and phosphorothiolates (withbridging sulfur atom), provides a foundationfor these and other experiments.

Nonenzymatic reactions have been charac-terized for a number of thio-substituted com-pounds and LFER and KIE data have beencollected (see Supplemental Sections E andF for data sets and references). As with phos-phate esters, these results are consistent withloose transition states for monoesters and pro-gressively tighter transition states for diesters



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and triesters. However, in contrast to phosphatemonoesters, which proceed through concertedreactions and do not form stable metaphosphateintermediates, thiometaphosphate intermedi-ates have been reported in some reactions ofphosphorothioates (79–81). This change can beunderstood in the context of a two-dimensionalreaction coordinate diagram, in which sul-fur substitution stabilizes the metaphosphatecorner sufficiently to allow formation of ametaphosphate intermediate (Figure 6). Thesimilarity in LFER trends for phosphorothioateand phosphate monoesters, diesters, and tri-esters, in combination with the observation offreely dissociated thiometaphosphate, providesfurther support for a loose, metaphosphate-like transition state for phosphate monoesterhydrolysis.

Overview of Nonenzymatic Data

The trends among all the data provide a con-sistent picture of the nature of the transi-tion state for phosphate monoesters, diesters,and triesters. Monoesters appear to progressthrough loose transition states, diesters throughmore synchronous transition states, and tri-esters through tighter transition states. In addi-tion, there is more variation in the nature of thetransition state for triesters than for monoestersin response to variation of the pKa values andreactivities of the attacking and leaving groups.This picture has not been challenged by newexperimental observations over several decades,yet each individual experiment is subject to un-certainties and the models must be continuouslyevaluated with respect to new data. Althoughcomputational methods fall outside of the scopeof this review, these approaches offer promisein more directly relating LFER and KIE datato transition-state structures and charge distri-butions. To establish confidence in the theo-retical models, systematic and comprehensiveexperimental data sets will be needed to serveas benchmarks, and computational approacheswill need to be critically assessed through robustand nontrivial predictions of previously unmea-sured values.

ENZYMATIC PHOSPHORYLTRANSFER

Phosphoryl-transfer reactions can be excep-tionally slow in solution (45, 82, 83), and en-zymes that catalyze these reactions can havetremendous rate accelerations. For example,the first-order rate constant for attack onmethyl phosphate dianion by water is less than10−20 s−1—a half-life of more than one trillionyears (45). Alkaline phosphatase catalyzes thishydrolysis with a second-order rate constant(kcat/KM) of 1.2 × 106 M−1s−1 (84), giving arate acceleration of more than 1027-fold!3

How do enzymes achieve these large rateaccelerations relative to the solution reactions?An understanding of the transition states fornonenzymatic reactions in solution can provideinsight into possible strategies for enzymaticcatalysis and can guide investigations of newenzymatic systems and catalytic mechanisms.In this section, we provide an overview ofstrategies that enzymes could employ incatalyzing phosphoryl-transfer reactions fromthe perspective of the nonenzymatic transitionstate, with an emphasis on monoesters. Earlierworks have reviewed possible catalytic strate-gies for phosphoryl transfer (e.g., 1, 85–88), andwe offer an updated perspective here, informedboth by our perspective on nonenzymatic reac-tions and by more recent work that sheds newlight on these strategies. Rather than coveringspecific enzyme classes and families, we referthe reader to other reviews (4–6, 89–93); wealso present three enzyme case studies in thesidebars and Supplemental Text that furtherillustrate specific mechanistic concepts.

Activation of the Nucleophile

Three general means of nucleophilic activationhave been proposed for reactions of phosphatemonoesters: positioning of the nucleophile,increasing nucleophilicity, and overcoming

3The second-order rate constant for the nonenzymatic reac-tion is the first-order rate constant divided by the concentra-tion of water (55 M): 3.6 × 10−22 M−1s−1.


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electrostatic repulsion. The current data, asoutlined below, suggest that only positioningcan provide substantial catalysis in reactionsproceeding via loose transition states, whereasincreasing the nucleophilicity through varyingthe identity of the nucleophile, metal ion catal-ysis, or general base catalysis can contributesignificantly for tighter transition states as indiesters and triesters.

Positioning. The most general hallmark ofenzymes is their ability to use binding interac-tions and positioned groups within the foldedenzyme to facilitate catalysis. This ability di-rectly links catalysis to specificity, as is requiredin biology (94). The ability to attain enormousrate advantages from positioning was demon-strated in many early studies that comparedinter- and intramolecular solution reactionsand was explained by Page & Jencks (95), whodemonstrated that such large effects couldcome from the removal of translational androtational degrees of freedom upon fixing tworeactants with respect to one another. Unfortu-nately, we still lack good estimates of how largesuch effects can be in enzymatic reactions (96).

In phosphoryl-transfer reactions, the nu-cleophile must be aligned with the phosphorusatom and the leaving group for in-line attackat phosphorus, and enzyme interactions couldhelp to position the nucleophile in this reactivegeometry. Empirical data for phosphoryl-transfer reactions suggest that positioningof the nucleophile can make a significantcontribution to catalysis. This conclusion isbased on exogenous rescue experiments inwhich the enzymatic His nucleophile of NDPkinase was replaced with noncovalently boundsmall-molecule nucleophiles. Comparisonsbetween the covalent enzymatic nucleophile,noncovalently bound nucleophiles, and thenonenzymatic reaction suggest a ∼102-fold en-hancement from positioning of the covalentlybound enzymatic nucleophile (97). How doesthis observation relate to our expectation thatthe transition state for phosphate monoesterhydrolysis has little nucleophilic participation?The simplest explanation is that even in a loose

transition state there is some bond formation tothe nucleophile, which requires positioning ofthe nucleophile with respect to the phosphorylgroup. Translational entropy is lost when thetwo reactants no longer move independentlyof one another, regardless of whether thetransition state is loose or tight, so a significantrate enhancement from prepositioning thereactants is possible.

Increasing the nucleophilicity. The reac-tivity of a nucleophile can be increased by anumber of mechanisms, including removalof a proton by a general base, activationby a metal ion, or by changing the identityof the nucleophile.4 The potential catalyticcontributions from general base catalysis ormetal ion activation can be estimated using theLFER slope βNUC, which describes the changein reaction rate as a function of nucleophilepKa. Qualitatively, because βNUC is small forreactions at monosubstituted phosphate cen-ters (Figure 5), there is little advantage gainedfrom changing the pKa of the nucleophilethrough any of these routes. Quantitatively, wecan calculate a maximum expected contributionusing the shift in pKa and the βNUC value.The expected log increase in rate constantupon abstraction of a proton from a water (orsimilarly serine) nucleophile is the difference inpKa between hydroxide (pKa = 15.7) and water(pKa = −1.7) times the βNUC value. Thus, theincrease in rate constant is estimated as

�(log k) = βNUC · �pKa.

For monoester reactions with βNUC = 0.1(Figure 5), this relationship gives a value of56-fold as the maximum rate increase, and fora hypothetical βNUC = 0.2, a value of 3 × 103-fold is obtained. The actual rate increase isexpected to be substantially less, as concerted

4For a given pKa, nitrogen nucleophiles are more reactive to-ward phosphorus than oxygen nucleophiles, while also form-ing thermodynamically weaker bonds. Thus, enzymes canobtain advantages from using nitrogen nucleophiles in cova-lent catalysis (98); such nucleophiles are used sometimes butnot exclusively.


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general base catalysis yields only partial depro-tonation in the transition state and thus partialactivation. There may be additional entropicbarriers for having multiple simultaneousevents of bond formation and proton transfer,further reducing the catalytic benefit for gen-eral base catalysis. Similar considerations applyto nucleophile activation by a metal ion. Theestimated pKa for the metal-bound alkoxideSer-O− · · · Zn2+ in the enzyme alkaline phos-phatase is ∼5.5 (99), giving a rate advantage of∼10-fold if βNUC ∼0.1–0.2.5 Overall, increas-ing nucleophilicity is not expected to providea major catalytic contribution for monoesterreactions with loose transition states. Thispoint is illustrated further in the sidebar RasGTPase: Is a General Base Necessary?

In contrast to the case for loose transitionstates, increasing the reactivity of the nucle-ophile through general base catalysis or metalion coordination can provide larger catalyticcontributions for reactions with tighter transi-tion states. As an example, for diester reactionswith βNUC = 0.31, the above relationship givesa value for the maximum increase in rate con-stant of 3 × 105, indicating that increasing nu-cleophilicity could provide a much larger con-tribution to catalysis for these tighter transitionstates, although again, general base catalysiswould not be expected to capture this full value.

Overcoming electrostatic repulsion. Couldan enzyme make phosphoryl transfer morefavorable by reducing electrostatic repulsionbetween the nucleophile and the chargedphosphoryl oxygen atoms? Westheimer, inhis classic perspective “Why Nature ChosePhosphates” (3), suggested that this type ofelectrostatic repulsion accounts for the lownonenzymatic reactivity of phosphate com-pounds. Indeed, nonenzymatic phosphatemonoester hydrolysis is extraordinarily slowrelative to other biological reactions (45). But is

5Here the metal-bound alkoxide species has a pKa signif-icantly lower than that of an alkoxide ion and is thus lessreactive than a free alkoxide.

RAS GTPASE: IS A GENERAL BASENECESSARY?

Ras and related proteins catalyze GTP hydrolysis and have beenstudied extensively as paradigms for molecular switches that linkenzymatic activity to cell-regulatory signals through conforma-tional change. The catalytic mechanism of the Ras GTPase be-came a topic of extensive discussion when structural studies didnot find a good candidate for a general base in the active site. Toaccount for this observation, a new mechanism was proposed,referred to as substrate-assisted catalysis, whereby the GTP sub-strate would act as the catalytic base to deprotonate water, form-ing the more potent hydroxide nucleophile (154, 155). Althoughthis mechanism is widely cited in the literature, subsequent ex-periments strongly support an alternative interpretation of theoriginal data taken as support of this mechanism; these exper-iments are described in Supplemental Section J. However, amore fundamental question underlies this issue: Is there any rea-son to think that a general base is necessary? Studies on nonenzy-matic and Mg2+-promoted nucleoside triphosphate hydrolysis aswell as Ras-catalyzed GTP hydrolysis suggest a loose transitionstate for this reaction (127, 138, 139). Using βNUC = 0.1 as re-ported for nucleoside triphosphate hydrolysis (127), the expectedcatalytic advantage for deprotonating water to form hydroxide isat most 60-fold, relative to a rate acceleration for Ras of approx-imately 105-fold relative to the nonenzymatic reaction. Thus,although interactions with the enzyme can help to position waterfor nucleophilic attack, there is little expected benefit from usinga general base to deprotonate the attacking water. Given consid-eration of the transition-state structure, additional mechanismsfor RasGAP-mediated GTP hydrolysis were proposed and sub-sequently supported by crystallographic and functional studies(discussed further in Supplemental Section J).

electrostatic repulsion the culprit? The obser-vation that no reaction of phosphate monoesterdianions with hydroxide ion is observed abovethat with water, even at high hydroxide ion con-centrations (20, 45), is consistent with the elec-trostatic repulsion model (100). However, analternative explanation is provided by the smallvalue of βNUC for these reactions (Figure 5). Asdiscussed above, with a βNUC value of 0.1, thereactivity of hydroxide ion can be roughly esti-mated as 60-fold greater than that of water. Butwater is present in 55-fold greater amounts even


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relative to 1 M hydroxide ion at pH 14. Thus,the absence of an observable rate increase athigh pH could primarily reflect the shallow de-pendence of the reaction rate on nucleophilicityand be unrelated to electrostatic repulsion.

A more direct experimental test of theelectrostatic repulsion model comes fromcomparisons of the reactivity of neutral andnegatively charged oxygen nucleophiles inreactions with negatively charged phosphateesters. These reactions all follow the samedependence on nucleophile pKa, with no signif-icant deviations between neutral and negativelycharged nucleophiles (33). Similarly, there areno deviations between charged and unchargednucleophiles when the rate constants for phos-phate ester hydrolysis are compared with thosefor corresponding reactions with uncharged es-ters (46). Finally, increasing the ionic strength,which would screen and thereby diminishelectrostatic repulsion, has only small effectson reaction rates; anionic nucleophiles react∼5-fold faster, whereas reactions of neutralnucleophiles are not significantly affected (46,101). These results suggest that electrostaticrepulsion between anionic nucleophiles andnegatively charged phosphate esters (relativeto uncharged esters) is not extensive.

If the relatively slow reactions of phosphateesters are not a consequence of electrostaticrepulsion between nucleophiles and negativelycharged phosphoryl groups, then mitigatingelectrostatic repulsion would not be a majorcontributor to enzymatic catalysis of thesereactions. However, further study of highlycharged phosphate esters such as nucleosidetriphosphates is warranted to further explorethis issue. For example, it has been suggestedfrom structural data that enzymes can bindATP with the phosphoryl oxygen atoms in aneclipsed conformation, introducing steric orelectrostatic repulsion that may destabilize thebound substrate and thereby lower the reactionbarrier (102, 103).

Stabilization of the Leaving Group

In contrast to the situation for the nucleophilediscussed above, the leaving group develops

substantial negative charge in the loose transi-tion state relative to the ground state for phos-phate monoester dianion reactions (Figure 3b).Consequently, stabilization of the developingnegative charge on the leaving group can con-tribute significantly to catalysis of monoesterreactions.

Similarly to the calculation above for nu-cleophilic activation, we can calculate a roughmaximum rate advantage for protonating analcohol-type leaving group such as glucose orethanol using the difference in pKa and the βLG

value with the following expression:

�(log k) = βLG · �pKa.

The maximum value of �pKa is the differencebetween the pKa of a fully protonated leavinggroup (pKa ∼ −2 for protonation of water or analcohol) and that for an alkoxide leaving group(pKa ∼ 16 for protonation of hydroxide and sim-ple alkoxides) (104). Using a value of �pKa of 18and the βLG value of −1.26 (Figure 5) gives anenormous maximum estimated rate advantageof >1020-fold. We emphasize that the actualexpected catalytic effect will be much less. Asnoted above for general base catalysis, the pro-ton will be partially rather than fully transferredin the transition state and there are entropic-like barriers for having multiple simultaneousevents of bond breaking and proton transfer.For phosphate diesters and triesters, the tightertransition states and correspondingly smallervalues of βLG lead to the prediction of smallercatalytic effects from a general acid, but largevalues of βLG (∼1) can still occur with a tighttransition state when βEQ is also large. In thesecases, general acid catalysis can provide a sig-nificant advantage.

Interactions with the PhosphorylOxygen Atoms: Charge, Positioning,and Geometry

The phosphoryl group (PO3−) that is trans-

ferred from the leaving group to the nu-cleophile can undergo two types of changesfrom the ground state to the transition state:


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electrostatic and geometric. The literature hasgenerally focused on electrostatic changes, butemerging data suggest that geometry may alsobe important.

Electrostatic changes. A hallmark of theactive sites of phosphoryl-transfer enzymes isthe presence of positively charged functionalgroups and metal ion cofactors in the activesite (1, 4, 91). These positively charged groupsare often positioned to interact with thenonbridging oxygen atoms of the transferredphosphoryl group. This observation has led tofrequent suggestions in the literature that thesepositively charged groups lead to enzymatictransition states that are tighter than those ofthe corresponding nonenzymatic reactions (1,28, 87, 91, 92, 102, 105–122). Most simply,coordination of the nonbridging oxygen atomscould make the substrate behave more like adiester or triester and thus proceed through atighter transition state.

A physical model for the proposed tight-ening of the transition state comes fromconsideration of electrostatic changes in thephosphoryl group. In a loose transition statewhere bond cleavage to the leaving group ismore advanced than bond formation to thenucleophile, there is a net loss of electrons inthe phosphoryl group and the negative chargeon the nonbridging oxygen atoms may decrease(Figure 9a). Indeed, to account for data sug-gesting extensive bond cleavage to the leavinggroup, early workers in the field suggested thatnegatively charged oxygen atoms provide thedriving force to expel the leaving group (18).In contrast, in a tighter transition state, the netincrease in bond order at phosphorus wouldbe expected to lead to an increase in negativecharge on the nonbridging phosphoryl oxygenatoms (Figure 9b). Thus, positively chargedfunctional groups that are ubiquitous inphosphoryl-transfer enzyme active sites havebeen suggested to be better suited to stabilizethe charge distribution in a tight transitionstate rather than a loose transition state similarto that in the nonenzymatic reaction. In termsof a two-dimensional reaction coordinate

–2/3

–2/3

–2/3

P

O

ORO

O

a Proposed charge distribution in a loose transition state

c Charge distribution in a metaphosphate-like species

P

O

O–O

b Proposed charge distribution in a tight transition state

Nu P

O

OR

OO

Nu+

P

O

ORO

ONu P

O

OR

OO

Nu+

P

O–

O–OP

O–

O––O

Increasing charge on

nonbridging oxygen atomsP

O

OOP

O

OO

+ +

–2/3

–2/3

–2/3

–1/3

–1/3 –1/3

–1

–1 –1

Figure 9Charge distribution on the phosphoryl oxygen atoms in the transition state.Based on a simple arrow-pushing scheme, one might expect the charge on thenonbridging oxygen atoms to decrease in a loose transition state (a) andincrease in a tight transition state (b) (the transition-state charges depicted arethe extreme limits). However, the charge distribution for a metaphosphate-likespecies in a loose transition state is unknown. Three different resonance formscan be depicted with more or less charge on the nonbridging oxygen atoms (c).

diagram, this proposal corresponds to anenzyme altering the energy surface relative tothat in solution to move the transition statefrom the bottom right of the diagram towardthe top left (Figure 6c, with positively chargedfunctional groups behaving like additionalester substituents on the phosphoryl group).

Although this logic is intuitively appealing,it falls short on several counts. First, the chargedistribution in a metaphosphate-like species ina loose transition state is not known, and there isno requirement that charge on the nonbridgingoxygen atoms should decrease to compensatefor leaving-group expulsion. Indeed, compu-tational studies of metaphosphate in the gasphase suggest a charge distribution closer tothe resonance forms on the right of Figure 9c,with significant negative charge density on thenonbridging oxygen atoms (123–125). Second,even if the charge on the nonbridging oxygen


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atoms does decrease in a loose transition state,positively charged functional groups in an en-zyme active site may form stronger interactionsin the transition state owing to changes in thegeometry of the phosphoryl group that leadto better alignment of the interacting groups.

ALKALINE PHOSPHATASE: DO POSITIVELYCHARGED GROUPS TIGHTENTRANSITION STATES?

Alkaline phosphatase (AP) catalyzes phosphoryl transfer from abroad range of monoesters and is among the most proficient en-zymes known. AP has considerable positive charge concentratedin its active site, including two Zn2+ ions and an arginine availableto interact with the nonbridging phosphoryl oxygen atoms in thetransition state. Could these interactions with positive chargelead to a tighter transition state, as may be expected from stabi-lization of the upper left corner in the two-dimensional reactioncoordinate diagram? Because the active site of AP accepts a widerange of leaving groups, it has been possible to perform numer-ous linear free energy relationship (LFER) and kinetic isotopeeffect studies to probe transition-state properties in AP-catalyzedreactions. In reactions of monoesters for which the chemicalstep is rate limiting, all experimental evidence, described in Sup-plemental Section I, is consistent with transition states that donot differ from those of the nonenzymatic reactions. These ob-servations suggest that the presence of positively charged groupsdoes not substantially tighten the transition state. Yet arginineresidues and other positively charged residues are ubiquitous inphosphoryl-transfer enzymes. What roles could they play? LFERresults showed no evidence for substantial change in transition-state structure upon mutation of the active-site arginine of AP(145), but measurement of individual reaction steps indicatedthat arginine coordination strengthens binding by approximately3 kcal/mol and provides 1–2 kcal/mol of additional transition-state stabilization (75). Through studies of AP and proteintyrosine phosphatase, another phosphoryl-transfer enzyme withsimilar arginine coordination in a very different active-site en-vironment, it has been proposed that interactions with arginineserve to position optimally the phosphoryl group for reactionand may provide further catalysis if they are positioned tointeract more strongly with the trigonal bipyramidal geometryin the transition state than the tetrahedral ground state (75, 76,115, 136, 156, 157). Additional references and discussion of APcatalytic mechanisms are provided in Supplemental Section I.

This idea is discussed further in the sidebarAlkaline Phosphatase: Do Positively ChargedGroups Tighten Transition States? Finally,the proposal that enzyme active sites alter thetransition state from that in solution has animportant unstated assumption: The energysurface surrounding the transition state mustbe sufficiently shallow such that the energeticbenefit gained from enzyme interactions witha tighter transition state outweighs the ener-getic preference for a loose transition state.Empirical studies to date suggest that these in-teractions are insufficient to overcome the pref-erence for a loose transition state for phosphatemonoester reactions. No evidence for changesin transition-state structure has been observedupon Mg2+ and Ca2+ coordination in reactionsof phosphate monoesters and anhydrides insolution, based on a lack of changes in LFERslopes, suggesting that these metal ion inter-actions are insufficient to alter transition-statestructure (126, 127).6 Consideration of the pxy

coefficient (see Supplemental Section C) alsosuggests that monoester transition states maynot be easy to change. Moreover, as experimen-tal LFER and KIE data have begun to emergefor enzyme-catalyzed phosphate monoesterreactions, changes in transition-state structurerelative to solution have also not generallybeen observed (56, 62, 99, 133–146). Phospho-glucomutase provides an interesting example,discussed in the sidebar Phosphoglucomutase:Do Enzymes Change Mechanisms fromThose in Solution?, of a case where enzymaticinteractions were proposed to cause extremetightening toward a phosphorane interme-diate, but follow-up investigations suggestedotherwise.

Thus, it appears unlikely that enzymesderive a catalytic advantage by shifting theenergy surface toward a tighter transition state

6Reactions of Co(III) complex phosphate esters provide anapparent exception (128–130). However, Co(III)-ligand in-teractions are generally very stable (131, 132). Thus, additionof Co(III) may mimic addition of a covalent substituent and,therefore, may be expected to result in a tighter transitionstate as does increased esterification of the phosphoryl group.


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and concurrently stabilizing greater chargedevelopment on the nonbridging oxygen atomsin the transition state. Nevertheless, furtherinvestigation is in order. Most fundamentally,the charge distribution of the phosphorylgroup in a loose transition state remains anopen question. A challenge for the future is todevelop experimental and computational toolsthat can confidently address this question.

The placement of active-site functionalgroups has also been suggested to determinethe distance between entering and leavinggroups in the transition state (92). This pro-posal makes assumptions about the nature ofthe energy surface for the reaction and howdifficult it is to change the transition-statestructure, and the structural data from X-ray crystallography and nuclear magnetic reso-nance provide information about ground states,not transition states. There is no indicationthat these distances can affect transition-statetightness.

Geometry and positioning. In nonenzymaticreactions, solvent must rearrange to accom-modate reaction transition states. In contrast,enzymatic interactions are optimally positionedby the folded macromolecule for electrostaticand geometric complementarity with the tran-sition state. These interactions include aspectsdiscussed above such as stabilizing contactsto the leaving group. In addition, interactionsbetween the nonbridging phosphoryl oxygenatoms and positively charged active-site groupscan play important catalytic roles in positioningthe phosphoryl substrate with respect to theattacking group and also in aligning the leavinggroup for stabilizing interactions. An open butintriguing question is whether the trigonalbipyramidal transition-state geometry can berecognized by the active site in preference tothe tetrahedral ground state. For phosphatemonoesters, the position and orientation of thenonbridging oxygen atoms changes in the pro-gression from ground state to transition state(Figure 3), and for diesters preferential inter-actions could also be made with the substituentattached to the transferred phosphoryl group.

PHOSPHOGLUCOMUTASE: DO ENZYMESCHANGE MECHANISMS FROM THOSEIN SOLUTION?

A recent X-ray crystallographic study challenged expectationsfrom nonenzymatic reactions by reporting a stable phosphoraneintermediate in phosphoryl transfer from glucose in the activesite of phosphoglucomutase (158). For this reaction, a concertedprocess with a loose transition state is expected, and thus the ob-servation of electron density suggestive of a stable pentacoordi-nate, trigonal bipyramidal species in the structure was surprising.A crystallographically observable phosphorane would representa remarkable shift in mechanism from a transition state near thelower right of the two-dimensional reaction coordinate diagramto a long-lived stable species in the upper left corner. However,soon after this result was presented, it was suggested that the ob-served pentacoordinate species may not represent a reaction in-termediate, but instead could represent a MgF3

− transition-stateanalog arising from components from the crystallization buffer(159). Subsequent evidence from 19F and 31P nuclear magneticresonance, additional X-ray experiments, and kinetic data pro-vided strong support for the assignment of the pentavalent speciesas MgF3

− (160, 161). The phosphoglucomutase story provides areminder of the importance of synergy between kinetics, chemi-cal mechanism, and structure for understanding enzymatic cataly-sis. Structural studies with transition-state analogs have advancedenzymology by providing physical models of active-site interac-tions. Nevertheless, structures alone cannot reveal mechanismor energetics and so must be placed in the context of functionalstudies. See Supplemental Section H for additional referencesand discussion.

This model implies that active-site interactionsare suboptimal in the ground state owing toconformational constraints and more favorablein the transition-state geometry. Thus, evenwith no increase in negative charge on thenonbridging oxygen atoms, hydrogen bondingand electrostatic interactions with these atomscould potentially become stronger in the tran-sition state as the nonbridging oxygen atomsreorient. Indeed, recent data support the possi-bility that enzymes can recognize small geomet-rical changes on the scale of a few tenths of anAngstrom (see Reference 147). Advancing ourunderstanding of enzyme rigidity, positioning,



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and dynamics is an important future challengefor phosphoryl transfer and other enzymes(96).

Determining the bond distances inphosphoryl-transfer transition states andtrajectories of reaction pathways representadditional important challenges. What arethe bond distances in loose or tight transitionstates, and do positioning interactions thatplace the reactants in close proximity lead totighter transition states? For example, it hasbeen suggested that the transition state canbe loose even if the nucleophile is initiallypositioned in van der Waals contact with thephosphate ester (5, 8).

Challenges for the Future

We can currently identify elements of enzy-matic systems that contribute to catalysis ofphosphoryl transfer, but ultimately we hope todevelop a quantitative, predictive understand-ing of the structural origins of catalysis. Onemajor challenge for achieving that goal is inrelating the empirical correlations providedby KIEs and LFERs to precise geometriesand charges for all atoms in the transitionstate. Both experimental and computationalapproaches toward this goal face additionallimitations caused by the complexity of the en-zymatic environment. Another major challengetoward a more quantitative understanding ofphosphoryl-transfer catalysis is in experimen-tally dissecting the roles of active-site interac-tions. Site-directed mutagenesis has providedcrucial information, but it cannot directlyassess catalytic contributions because activesites are complex and cooperative networks of

interacting residues (96). Double-mutantcycles can help by assessing cooperativity ofinteractions, albeit imperfectly (148, 149).Comparative approaches that utilize multiplesubstrates, multiple enzymes, and catalyticpromiscuity to further parse energetic effectsare providing additional insights (84, 150–153).Yet understanding energetic contributionsremains difficult, and much work remains tobe done.

CONCLUSIONS

Phosphoryl-transfer enzymes have attractedenormous attention as befits their central rolesin biology. Over time, different approaches tothese systems have been prominent: physicalorganic approaches to nonenzymatic phospho-ryl transfer in the 1950s, mechanistic studiesof specific enzymes in the 1970s and beyond,structural studies from the 1990s to the present,and contemporary emerging computationalapproaches. Although each new developmenthas been powerful and exciting, there has beena tendency to lose track of mechanistic con-siderations from earlier nonenzymatic studies.We hope that this review will resuscitateand broaden a fundamental understandingof phosphoryl-transfer mechanisms and thatthe analyses and case studies will providemotivation for current and future investiga-tors to apply lessons and approaches fromnonenzymatic studies. The understandingof biological phosphoryl transfer from theseapproaches is synergistic rather than additive,and the deepest understanding will come froman integration of different perspectives andmethods.

SUMMARY POINTS

1. Considerable experimental evidence supports a concerted reaction for phosphate mo-noesters and related compounds in nearly all cases.

2. Transition states for phosphoryl-transfer reactions can, in principle, be loose, tight, orsynchronous, as defined by the net bonding of phosphorus to the nucleophile and leavinggroup.


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3. A consistent picture of the transition states for phosphoryl transfer has emerged fromLFER and KIE studies, where phosphate monoesters proceed through loose transitionstates, phosphate diesters through roughly synchronous transition states, and phosphatetriesters through generally tighter but more variable transition states.

4. Experimental evidence suggests that, in cases tested to date, enzymatic catalysis generallydoes not substantially alter transition states for phosphoryl transfer from those in solution.

5. Consideration of the mechanistic data for nonenzymatic phosphoryl transfer allows eval-uation of proposed catalytic mechanisms and has led to important insights into mecha-nisms of catalysis for enzymatic phosphoryl transfer.

FUTURE ISSUES

1. LFER and KIE data provide empirical descriptions of transition states. A major challengelies in translating the empirical correlations into bond distances, electronic distributions,and vibrational states.

2. Additional, systematic experimental data will be needed to achieve this goal. Compu-tational approaches offer promise in correlating these empirical data with physical pa-rameters, but calculations must be related to these experimental observables and providenontrivial predictions to allow unbiased tests of the methods and results.

3. Understanding how reaction energy surfaces change in response to enzymatic stabilizinginteractions is a key challenge. An ultimate goal is the development of predictive andquantitative models for phosphoryl-transfer catalysis.

4. Although not discussed here, a related important challenge is to understand how enzy-matic catalysis of phosphoryl transfer is coupled to events required for biological signalingand regulation.

DISCLOSURE STATEMENT

The authors are not aware of any biases that might be perceived as affecting the objectivity of thisreview.

ACKNOWLEDGMENTS

This work was supported by a grant from the National Institutes of Health (NIH grant numberGM64798 to D.H.). J.K.L was supported by a postdoctoral fellowship from the NIH (GM080865).J.G.Z. was supported by the Damon Runyon Cancer Research Foundation. For helpful discussionsand comments, we thank Alvan Hengge, Nick Williams, and members of the Herschlag lab.

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BI80-FrontMatter ARI 16 May 2011 14:13

Annual Review ofBiochemistry

Volume 80, 2011Contents

Preface

Past, Present, and Future Triumphs of BiochemistryJoAnne Stubbe � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �v

Prefatory

From Serendipity to TherapyElizabeth F. Neufeld � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 1

Journey of a Molecular BiologistMasayasu Nomura � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �16

My Life with NatureJulius Adler � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �42

Membrane Vesicle Theme

Protein Folding and Modification in the MammalianEndoplasmic ReticulumIneke Braakman and Neil J. Bulleid � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �71

Mechanisms of Membrane Curvature SensingBruno Antonny � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 101

Biogenesis and Cargo Selectivity of AutophagosomesHilla Weidberg, Elena Shvets, and Zvulun Elazar � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 125

Membrane Protein Folding and Insertion Theme

Introduction to Theme “Membrane Protein Folding and Insertion”Gunnar von Heijne � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 157

Assembly of Bacterial Inner Membrane ProteinsRoss E. Dalbey, Peng Wang, and Andreas Kuhn � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 161

β-Barrel Membrane Protein Assembly by the Bam ComplexChristine L. Hagan, Thomas J. Silhavy, and Daniel Kahne � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 189

vii

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Transmembrane Communication: General Principles and Lessonsfrom the Structure and Function of the M2 Proton Channel, K+

Channels, and Integrin ReceptorsGevorg Grigoryan, David T. Moore, and William F. DeGrado � � � � � � � � � � � � � � � � � � � � � � � � 211

Biological Mass Spectrometry Theme

Mass Spectrometry in the Postgenomic EraBrian T. Chait � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 239

Advances in the Mass Spectrometry of Membrane Proteins:From Individual Proteins to Intact ComplexesNelson P. Barrera and Carol V. Robinson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 247

Quantitative, High-Resolution Proteomics for Data-DrivenSystems BiologyJurgen Cox and Matthias Mann � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 273

Applications of Mass Spectrometry to Lipids and MembranesRichard Harkewicz and Edward A. Dennis � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 301

Cellular Imaging Theme

Emerging In Vivo Analyses of Cell Function UsingFluorescence ImagingJennifer Lippincott-Schwartz � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 327

Biochemistry of Mobile Zinc and Nitric Oxide Revealedby Fluorescent SensorsMichael D. Pluth, Elisa Tomat, and Stephen J. Lippard � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 333

Development of Probes for Cellular Functions Using FluorescentProteins and Fluorescence Resonance Energy TransferAtsushi Miyawaki � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 357

Reporting from the Field: Genetically Encoded Fluorescent ReportersUncover Signaling Dynamics in Living Biological SystemsSohum Mehta and Jin Zhang � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 375

Recent Advances in Biochemistry

DNA Replicases from a Bacterial PerspectiveCharles S. McHenry � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 403

Genomic and Biochemical Insights into the Specificity of ETSTranscription FactorsPeter C. Hollenhorst, Lawrence P. McIntosh, and Barbara J. Graves � � � � � � � � � � � � � � � � � � � 437

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Signals and Combinatorial Functions of Histone ModificationsTamaki Suganuma and Jerry L. Workman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 473

Assembly of Bacterial RibosomesZahra Shajani, Michael T. Sykes, and James R. Williamson � � � � � � � � � � � � � � � � � � � � � � � � � � � � 501

The Mechanism of Peptidyl Transfer Catalysis by the RibosomeEdward Ki Yun Leung, Nikolai Suslov, Nicole Tuttle, Raghuvir Sengupta,

and Joseph Anthony Piccirilli � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 527

Amyloid Structure: Conformational Diversity and ConsequencesBrandon H. Toyama and Jonathan S. Weissman � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 557

AAA+ Proteases: ATP-Fueled Machines of Protein DestructionRobert T. Sauer and Tania A. Baker � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 587

The Structure of the Nuclear Pore ComplexAndre Hoelz, Erik W. Debler, and Gunter Blobel � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 613

Benchmark Reaction Rates, the Stability of Biological Moleculesin Water, and the Evolution of Catalytic Power in EnzymesRichard Wolfenden � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 645

Biological Phosphoryl-Transfer Reactions: Understanding Mechanismand CatalysisJonathan K. Lassila, Jesse G. Zalatan, and Daniel Herschlag � � � � � � � � � � � � � � � � � � � � � � � � � � � 669

Enzymatic Transition States, Transition-State Analogs, Dynamics,Thermodynamics, and LifetimesVern L. Schramm � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 703

Class I Ribonucleotide Reductases: Metallocofactor Assemblyand Repair In Vitro and In VivoJoseph A. Cotruvo Jr. and JoAnne Stubbe � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 733

The Evolution of Protein Kinase Inhibitors from Antagoniststo Agonists of Cellular SignalingArvin C. Dar and Kevan M. Shokat � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 769

Glycan Microarrays for Decoding the GlycomeCory D. Rillahan and James C. Paulson � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 797

Cross Talk Between O-GlcNAcylation and Phosphorylation:Roles in Signaling, Transcription, and Chronic DiseaseGerald W. Hart, Chad Slawson, Genaro Ramirez-Correa, and Olof Lagerlof � � � � � � � � � 825

Regulation of Phospholipid Synthesis in the YeastSaccharomyces cerevisiaeGeorge M. Carman and Gil-Soo Han � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 859

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Sterol Regulation of Metabolism, Homeostasis, and DevelopmentJoshua Wollam and Adam Antebi � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 885

Structural Biology of the Toll-Like Receptor FamilyJin Young Kang and Jie-Oh Lee � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 917

Structure-Function Relationships of the G Domain, a CanonicalSwitch MotifAlfred Wittinghofer and Ingrid R. Vetter � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 943

STIM Proteins and the Endoplasmic Reticulum-PlasmaMembrane JunctionsSilvia Carrasco and Tobias Meyer � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � 973

Amino Acid Signaling in TOR ActivationJoungmok Kim and Kun-Liang Guan � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1001

Mitochondrial tRNA Import and Its Consequencesfor Mitochondrial TranslationAndre Schneider � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1033

Caspase Substrates and Cellular RemodelingEmily D. Crawford and James A. Wells � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1055

Regulation of HSF1 Function in the Heat Stress Response:Implications in Aging and DiseaseJulius Anckar and Lea Sistonen � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1089

Indexes

Cumulative Index of Contributing Authors, Volumes 76–80 � � � � � � � � � � � � � � � � � � � � � � � � � �1117

Cumulative Index of Chapter Titles, Volumes 76–80 � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � � �1121

Errata

An online log of corrections to Annual Review of Biochemistry articles may be found athttp://biochem.annualreviews.org/errata.shtml

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Biological Phosphoryl-Transfer Reactions: Understanding

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